Gene transfer of tumor necrosis factor inhibitor improves the function of lung allografts

BACKGROUND: Tumor necrosis factor is an important mediator of lung transplant acute rejection. Soluble type I tumor necrosis factor receptor binds to tumor necrosis factor-alpha and -beta and inhibits their function. The objectives of this study were to demonstrate efficient in vivo gene transfer of a soluble type I tumor necrosis factor receptor fusion protein (sTNF-RI-Ig) and determine its effects on lung allograft acute rejection. METHODS: Three groups of Fischer rats (n = 6 per group) underwent recipient intramuscular transfection 24 hours before transplantation with saline, 1 x 10(10) plaque-forming units of control adenovirus encoding beta-galactosidase, or 1 x 10(10) plaque-forming units of adenovirus encoding human sTNF-RI-Ig (Ad.sTNF-RI-Ig). One group (n = 6) received recipient intramuscular transfection with 1 x 10(10) Ad.sTNF-RI-Ig at the time of transplantation. Brown Norway donor lung grafts were stored for 5 hours before orthotopic lung transplantation. Graft function and rejection scores were assessed 5 days after transplantation. Time-dependent transgene expression in muscle, serum, and lung grafts were evaluated by using enzyme-linked immunosorbent assay of human soluble type I tumor necrosis factor receptor. RESULTS: Recipient intramuscular transfection with 1 x 10(10) plaque-forming units of Ad.sTNF-RI-Ig significantly improved arterial oxygenation when delivered 24 hours before transplantation compared with saline, beta-galactosidase, and Ad.sTNF-RI-Ig transfection at the time of transplantation (435.8 +/- 106.6 mm Hg vs 142.3 +/- 146.3 mm Hg, 177.4 +/- 153.7 mm Hg, and 237.3 +/- 185.2 mm Hg; P =.002,.005, and.046, respectively). Transgene expression was time dependent, and there was a trend toward lower vascular rejection scores (P =.066) in the Ad.sTNF-RI-Ig group transfected 24 hours before transplantation. CONCLUSIONS: Recipient intramuscular Ad.sTNF-RI-Ig gene transfer improves allograft function in a well-established model of acute rejection. Maximum benefit was observed when transfection occurred 24 hours before transplantation.     

Tumor necrosis factor inhibitor gene transfer ameliorates lung graft ischemia-reperfusion injury

OBJECTIVE: Tumor necrosis factor is an important mediator of lung transplant ischemia-reperfusion injury, and soluble type I tumor necrosis factor receptor binds to tumor necrosis factor and works as a tumor necrosis factor inhibitor. The objectives of this study were to demonstrate that gene transfer of type I tumor necrosis factor receptor-IgG fusion protein reduces lung isograft ischemia-reperfusion injury and to compare donor endobronchial versus recipient intramuscular transfection strategies. METHODS: Three donor groups of Fischer rats (n = 6/group) underwent endobronchial transfection with either saline, 2 x 10(7) plaque-forming units of control adenovirus encoding beta-galactosidase, or 2 x 10(7) plaque-forming units of adenovirus encoding type I tumor necrosis factor receptor-IgG fusion protein. Left lungs were harvested 24 hours later. Two recipient groups (n = 6/group) underwent intramuscular transfection with 2 x 10(7) plaque-forming units or 1 x 10(10) plaque-forming units of adenovirus encoding type I tumor necrosis factor receptor-IgG fusion protein 24 hours before transplantation. All donor lung grafts were stored for 18 hours before orthotopic lung transplantation. Graft function was assessed 24 hours after reperfusion. Transgene expression was evaluated by means of enzyme-linked immunosorbent assay and immunohistochemistry of type I tumor necrosis factor receptor. RESULTS: Endobronchial transfection of donor lung grafts with 2 x 10(7) plaque-forming units of adenovirus encoding type I tumor necrosis factor receptor-IgG fusion protein significantly improved arterial oxygenation compared with the saline and beta-galactosidase donor groups (366.6 +/- 137.9 vs 138.8 +/- 159.9 and 140.6 +/- 131.4 mm Hg, P =.009 and.010, respectively). Recipient intramuscular transfection with 1 x 10(10) plaque-forming units of adenovirus encoding type I tumor necrosis factor receptor-IgG fusion protein improved lung graft oxygenation compared with that seen in the low-dose intramuscular group (2 x 10(7); 320.3 +/- 188.6 vs 143.6 +/- 20.2 mm Hg, P =.038). Type I tumor necrosis factor receptor-IgG fusion protein was expressed in endobronchial transfected grafts. In addition, intramuscular type I tumor necrosis factor receptor-IgG fusion protein expression was dose dependent. CONCLUSIONS: Donor endobronchial and recipient intramuscular adenovirus-mediated gene transfer of type I tumor necrosis factor receptor-IgG fusion protein improved experimental lung graft oxygenation after prolonged ischemia. However, donor endobronchial transfection required 500-fold less vector. Furthermore, at low vector doses, it does not create significant graft inflammation.     

Recipient intramuscular cotransfection of transforming growth factor beta1 and interleukin 10 ameliorates acute lung graft rejection

OBJECTIVE: Multiple gene transfer might permit modulation of concurrent biochemical pathways involved in acute lung graft rejection. We investigated whether gene cotransfection into the recipient reduces acute lung graft rejection. METHODS: Brown Norway rats were used as donors, and F344 rats were used as recipients. Recipient animals were injected with saline (groups I/VI) or 1 x 10(10) pfu of adenovirus encoding beta-galactosidase (groups II/VII), transforming growth factor beta1 (groups III/VIII), interleukin 10 (groups IV/IX), or both transforming growth factor beta1 and interleukin 10 (groups V/X) into both leg muscles 2 days before transplantation (groups I-V) or at the time of harvest (groups VI-X). The Kruskal-Wallis test for rejection score and 1-way analysis of variance were used to compare groups. RESULTS: Oxygenation was significantly improved in the cotransfected groups treated 2 days before transplantation and at the time of harvest. Rejection scores were also reduced in the cotransfected groups. In group V cotransfection suppressed endogenous interleukin 2 but not interferon gamma and tumor necrosis factor alpha. CONCLUSION: Recipient intramuscular cotransfection of transforming growth factor beta1 and interleukin 10 suppressed interleukin 2 expression and provided a synergistic effect that reduced acute lung graft rejection. This approach might be applied to the clinical setting because transplant recipients could be treated at the time of implantation.     

Ex vivo transfection of transforming growth factor-beta1 gene to pulmonary artery segments in lung grafts

OBJECTIVE: Proximal pulmonary artery segment transfection may provide beneficial downstream effects on the whole-lung graft. In this study, transforming growth factor-beta1 was transfected to proximal pulmonary artery segments, and the efficacy of transforming growth factor-beta1 transfection was examined in ischemia-reperfusion injury and acute rejection models of rat lung transplantation. METHODS: In the ischemia-reperfusion injury model, orthotopic left lung transplantation was performed in F344 rats. In group I, the PPAS was isolated and injected with saline solution. In 2 other groups, lipid67:DOPE:sense (group II) or antisense transforming growth factor-beta1pDNA construct (group III) was injected instead of saline solution. After cold preservation at 4 degrees C for 18 hours, lung grafts were implanted. Graft function was assessed 24 hours later. In the acute rejection model, donor lung grafts were harvested. Proximal pulmonary artery segments were injected with saline solution (group I) or sense (group II) or antisense lipid gene construct (group III) and then implanted. Graft function was assessed on postoperative day 5. RESULTS: In the ischemia-reperfusion injury study, there were no significant differences in oxygenation, wet-to-dry weight ratios, graft myeloperoxidase activity, or transforming growth factor-beta1 levels in platelet-poor serum or proximal pulmonary artery segment homogenates. In the acute rejection study, oxygenation was significantly improved in group II receiving transforming growth factor-beta1 (group II vs I and III, 136.0 +/- 32.5 vs 54.0 +/- 9.6 mm Hg and 53.8 +/- 14.8 mm Hg; P =.016 and.016). There were no significant pathologic differences. Transforming growth factor-beta1 concentrations from proximal pulmonary artery segment homogenates in group II were significantly higher compared with controls. CONCLUSIONS: Ex vivo transfection of transforming growth factor-beta1 to proximal pulmonary artery segments did not affect reperfusion injury of lung isografts. In acute rejection, however, ex vivo transfection of transforming growth factor-beta 1 to proximal pulmonary artery segments improved allograft function. This suggests that transfection to proximal pulmonary artery segments exerts beneficial downstream effects on the whole-lung allograft.     

Transforming growth factor-beta1 gene transfer ameliorates acute lung allograft rejection

BACKGROUND: The aim of the current work was to study the feasibility of functional gene transfer using the gene encoding for transforming growth factor-beta1, a known immunosuppressive cytokine, on rat lung allograft function in the setting of acute rejection. METHODS: The rat left lung transplant technique was used in all experiments, with Brown Norway donor rats and Fischer recipient rats. After harvest, left lungs were transfected ex vivo with either sense or antisense transforming growth factor-beta1 constructs complexed to cationic lipids, then implanted into recipients. On postoperative days 2, 5, and 7, animals were put to death, arterial oxygenation measured, and acute rejection graded histologically. RESULTS: On postoperative day 2, there were no differences in acute rejection or lung function between animals treated with transforming growth factor-beta1 and control animals. On postoperative day 5, oxygenation was significantly improved in grafts transfected with the transforming growth factor-beta1 sense construct compared with antisense controls (arterial oxygen tension = 411 +/- 198 vs 103 +/- 85 mm Hg, respectively; P =.002). Acute rejection scores from lung allografts were also significantly improved, corresponding to decreases in both vascular and airway rejection (vascular rejection scores: 2.0 +/- 0. 5 vs 2.8 +/- 0.6; P =.04; airway rejection scores: 1.3 +/- 0.7 vs 2. 3 +/- 0.8, respectively; P =.02). The amelioration of acute rejection was temporary and decreased by postoperative day 7. CONCLUSIONS: The feasibility of using gene transfer techniques to introduce novel functional genes in the setting of lung transplantation is demonstrated. In this model of rat lung allograft rejection, gene transfer of transforming growth factor-beta1 resulted in temporary but significant improvements in lung allograft function and acute rejection pathology.     

Recipient intramuscular cotransfection of naked plasmid transforming growth factor beta1 and interleukin 10 ameliorates lung graft ischemia-reperfusion injury

OBJECTIVE: Multiple gene transfer might permit modulation of concurrent biochemical pathways involved in lung graft ischemia-reperfusion injury. In this study we analyzed whether recipient intramuscular naked plasmid cotransfection of transforming growth factor beta(1) and interleukin 10 would result in amelioration of lung graft ischemia-reperfusion injury. METHODS: Forty-eight hours before transplantation, 6 groups (n = 6) of F344 rats received intramuscular injection of naked plasmid encoding chloramphenicol acetyltransferase, chloramphenicol acetyltransferase plus beta-galactosidase, transforming growth factor beta(1), interleukin 10, or transforming growth factor beta(1) plus interleukin 10 or were not treated. Donor lungs were flushed and stored for 18 hours at 4 degrees C before transplantation. Twenty-four hours later, grafts were assessed immediately before the animals were killed. Arterial oxygenation, wet/dry ratio, myeloperoxidase, and proinflammatory cytokines (interleukin 1, tumor necrosis factor alpha, interferon gamma, and interleukin 2) were measured, and immunohistochemistry was performed. RESULTS: For lung graft function, the arterial oxygenation was considerably higher in the cotransfected group receiving transforming growth factor beta(1) plus interleukin 10 compared with that in all other groups (P < or =.03). The wet/dry ratio, reflecting lung edema, was reduced in the cotransfected group compared with that in control animals (nontreated, P <.02; chloramphenicol acetyltransferase, P <.03; chloramphenicol acetyltransferase plus beta-galactosidase, P <.01). Myeloperoxidase, which measures neutrophil sequestration, was also reduced with cotransfection compared with that seen in control animals (P < or =.03). All proinflammatory cytokines were decreased in the cotransfected group compared with those in all other groups (interleukin 1beta, P <.04; tumor necrosis factor alpha, P <.002; interferon gamma, P <.0001; interleukin 2, P <.03). These results indicate that cotransfection provides a synergistic benefit in graft function versus either cytokine alone, neutrophil sequestration, or inflammatory cytokine expression. Immunohistochemistry showed positive staining of transforming growth factor beta(1) plus interleukin 10 in type I and II pneumocytes and localized edema fluid. CONCLUSIONS: Recipient intramuscular naked plasmid cotransfection of transforming growth factor beta(1) and interleukin 10 provides a synergistic effect in ameliorating lung reperfusion injury after prolonged ischemia.     

Modulation of the biologic activity of the rabbit intervertebral disc by gene therapy: an in vivo study of adenovirus-mediated transfer of the human transforming growth factor beta 1 encoding gene

STUDY DESIGN: In vivo studies using a rabbit model to determine the biologic effects of direct, adenovirus-mediated transfer of a therapeutic gene to the intervertebral disc. OBJECTIVES: 1) To deliver an exogenous therapeutic gene to rabbit lumbar intervertebral discs in vivo, 2) to quantify the resulting amount of gene expression, and 3) to determine the effect on the biologic activity of the discs. SUMMARY OF BACKGROUND DATA: Although growth factors such as transforming growth factor beta 1 appear to have promising therapeutic properties, there currently is no practical method for sustained delivery of exogenous growth factors to the disc for the management of certain chronic types of disease (e.g., disc degeneration). A possible solution is to modify the disc cells genetically through gene transfer such that the cells manufacture the desired growth factors endogenously on a continuous basis. METHODS: Saline, with or without virus, was injected directly into lumbar discs of 22 skeletally mature female New Zealand white rabbits. Group 1 (n = 11) received the adenovirus construct Ad/CMV-hTGF beta 1 containing the therapeutic human transforming growth factor beta 1-encoding gene. Group 2 (n = 6) received adenovirus containing the luciferase marker gene. Group 3 (n = 5) received saline only. The rabbits were killed 1 week after injection. Immunohistochemical staining for human transforming growth factor beta 1 was performed on the disc tissues of one rabbit from Group 1. Nucleus pulposus tissues from the remaining rabbits were cultured in serumless medium. Bioassays were performed to determine human transforming growth factor beta 1 production and proteoglycan synthesis. RESULTS: Discs injected with Ad/CMV-hTGF beta 1 exhibited extensive and intense positive immunostaining for transforming growth factor beta 1. The nucleus pulposus tissues from the discs injected with Ad/CMV-hTGF beta 1 exhibited a 30-fold increase in active transforming growth factor beta 1 production, and a 5-fold increase in total (active + latent) transforming growth factor beta 1 production over that from intact control discs (P < 0.05). Furthermore, these tissues exhibited a 100% increase in proteoglycan synthesis compared with intact control tissue, which was statistically significant (P < 0.05). CONCLUSIONS: The results of this study suggest that the intervertebral disc is an appropriate site for adenovirus-mediated transfer of exogenous genes and subsequent production of therapeutic growth factors. Gene therapy therefore may have useful applications for study of the basic science of the intervertebral disc and for clinical management of degenerative disc disease.     

Low-dose endobronchial gene transfer to ameliorate lung graft ischemia-reperfusion injury

OBJECTIVE: This study was undertaken to determine whether low-dose endobronchial transfer to the donor of the gene for human interleukin 10 would decrease ischemia-reperfusion injury in lung transplantation. METHODS: Experiments used male Fischer rats. Donor animals underwent right thoracotomy. A catheter was introduced into the left main bronchus, and vector was instilled. Group I (n = 6) received 2 x 10(7) plaque-forming units of adenovirus encoding human interleukin 10, group II (n = 6) received an adenovirus control encoding beta-galactosidase, and group III (n = 6) received saline solution. After instillation the left main bronchus was clamped for 60 minutes. Lungs were removed 24 hours later and stored in low-potassium dextran glucose solution for 18 hours before left lung transplantation. Graft function was assessed at 24 hours immediately before the animals were killed. Ratio of wet to dry weight and tissue myeloperoxidase activity were measured. Transgenic expression of human interleukin 10 was evaluated by means of enzyme-linked immunosorbent assay and immunohistochemical assay. RESULTS: Arterial oxygenation was significantly improved in group I relative to groups II and III (257.6 +/- 59.7 mm Hg vs 114.6 +/- 66.9 mm Hg and 118.6 +/- 91.1 mm Hg, P =.008 and P =.007, respectively). Neutrophil sequestration, as measured by myeloperoxidase activity, was also significantly reduced in group I relative to groups II and III (0.141 +/- 0.025 vs 0.304 +/- 0.130 and 0.367 +/- 0.153 Delta optical density units/[min. mg protein], P =.029 and P =.004, respectively). Enzyme-linked immunosorbent assay and immunohistochemical assay demonstrated the expression of human interleukin 10 in transfected lungs only. CONCLUSIONS: Low-dose endobronchial transfer to the donor of the gene for human interleukin 10 ameliorated ischemia-reperfusion injury in rodent lung transplantation by improving graft oxygenation and reducing neutrophil sequestration. Only 2 x 10(7) plaque-forming units of adenoviral vector were required for functional transgenic expression. Endobronchial gene transfer to lung grafts may be a useful delivery route even at low doses.     

Application of HVJ-liposome mediated gene transfer in lung transplantation-distribution and transfection efficiency in the lung.

OBJECTIVE: A novel hemagglutinating virus of Japan (HVJ)-liposome-mediated gene transfer system has been shown to have benefits of a high efficiency of transfection and low immunogenicity. The aims of this study were to determine the effect of re-transfection of the HVJ-liposome system via the airway, and to quantify the distribution of gene expression between transtracheal and transplantation approaches. METHODS: Beta-galactosidase (beta-gal) plasmid DNA was introduced into lung tissues using the HVJ-liposome method. Two groups of Sprague-Dawley (SD) rats received intratracheal instillation of 10 microg of the beta-gal gene, once on Day 0 in 1 group (Group Tb-1, n=4) and 3 times on Days 0-2 in another (Group Tb-3, n=4). In a third group of SD rats (Group Tx, n=5), an orthotopic left lung transplantation was performed after the donor lung was flushed with an HVJ-liposome complex solution and preserved for 1h. Gene expression and distribution in lung tissue was then quantified by counting the X-gal stained cells. RESULTS: Both the transtracheal and transplantation approaches resulted in low levels of transfection in the vascular endothelial cells (0.2+/-0.1 and 4.0+/-1.8%), respectively, but a moderate degree of transfection to the airway (11.0+/-7.1 and 28.0+/-20.7%) and alveolar cells (3.0+/-1.8 and 6.0+/-3.6%). Three repetitive injections via the airway increased gene expression in airway epithelial cells of 41.0+/-12.0% compared with the single administration of 11.0+/-4.3%. CONCLUSIONS: Our results suggest that the repeated transtracheal gene transfection using HVJ-liposome may have benefits for treatment of problems after lung transplantation. In addition, gene transfer using a flushing solution during harvest may provide an opportunity for gene manipulation in the setting of lung transplantation.     

Intratracheal adenovirus-mediated gene transfer is optimal in experimental lung transplantation

OBJECTIVE: Gene transfer to experimental lung grafts has been shown to reduce ischemia-reperfusion injury and acute rejection. The optimal delivery route should produce high lung expression with no inflammation and minimal systemic expression. The goal of this study was to determine the optimal gene transfer route for use in experimental lung transplantation. METHODS: F344 rats were injected with 2.9 x 10(10) plaque-forming units of adenovirus vector encoding beta-galactosidase through intratracheal, intravenous, intraperitoneal, or intramuscular delivery routes and killed 48 hours later. Gene expression was measured by means of enzyme-linked immunosorbent assay. RESULTS: Intratracheal delivery produces significantly greater gene expression in the lung (75,350 +/- 47,288 pg/100 microg of protein, P <.001 vs intravenous, intraperitoneal, and intramuscular routes) and minimal systemic expression (nonsignificant in serum, kidney, liver, spleen, and muscle vs that seen in control animals, P =.016 for heart). Immunohistochemistry staining showed beta-galactosidase expression in the bronchial epithelium of lungs transfected through the intratracheal route with mild inflammation. CONCLUSIONS: Intratracheal gene transfer provides significant expression in the lung with mild to no inflammation and minimal systemic expression. This delivery strategy has tremendous potential in experimental lung transplant models to reduce ischemia-reperfusion injury and acute allograft rejection and should be investigated further.     

Intramuscular Gene Transfer of Interleukin-10 Reduces Neutrophil Recruitment and Ameliorates Lung Graft Ischemia-Reperfusion Injury

Interleukin-10 (IL-10) has potent anti-inflammatory properties but its direct effects on neutrophil trafficking in lung transplant ischemia-reperfusion (I/R) injury are unknown. This study was performed to determine if recipient intramuscular IL-10 gene transfer reduces neutrophil infiltration in lung isografts and ameliorates I/R injury. Twenty-four hours before transplantation, recipient rodents received intramuscular injection with 1 x 10(10) plaque-forming units (pfu) adenovirus encoding human IL-10 (hIL-10), 1 x 10(10) pfu adenovirus control encoding p-galactosidase, or saline. Gene expression in muscle and plasma was confirmed. Lung grafts were harvested, stored at 4 degrees C for 18h, and assessed 24 h after transplantation. Peak muscle and plasma expression of hIL-10 was achieved 24h after gene transfer and returned to baseline by 7 days (p < 0.05 vs. controls). Gene transfer of hIL-10 reduced neutrophil sequestration and emigration in lung grafts as measured by morphometry and myeloperoxidase activity (p < 0.03 vs. controls). Furthermore, hIL-10 improved graft oxygenation and reduced lung edema (p <0.01 vs. controls). Intramuscular gene transfer of hIL-10 releases hIL-10 protein into plasma and reduces neutrophil sequestration and emigration in lung isografts. This is associated with a reduction in I/R injury with improved isograft oxygenation and reduced tissue edema. Intramuscular gene transfer may be a useful strategy to reduce clinical l/R injury.     

Recipient intramuscular administration of naked plasmid TGF-β1 attenuates lung graft reperfusion injury

BACKGROUND: Gene therapy may be an effective strategy for modulating lung graft ischemia-reperfusion injury. We investigated whether recipient intramuscular (IM) naked plasmid gene transfer of transforming growth factor beta1-active (TGF-beta1-active) ameliorates lung graft ischemia-reperfusion injury. METHODS: Preliminary studies in F344 rats demonstrated that gastrocnemius muscle transfection of TGF-beta1-active produced muscle and plasma protein expression at 24 and 48 hours after transfection. Recipients (n = 8) received IM injection of naked plasmid-encoding chloramphenicol acetyl transferase (CAT), TGF-beta1-latent or TGF-beta1-active, respectively, at 24 or at 48 hours before left lung transplantation. We did not treat the control group before transplantation (18-hour cold ischemia). Donor lungs were flushed with low-potassium dextran-1% glucose and stored for 18 hours at 4 degrees C. All groups were killed at 24 hours after transplantation. Immediately before killing the animals, we clamped the contralateral right hilum and assessed graft function. We measured wet-to-dry ratio (W/D), myeloperoxidase, pro-inflammatory cytokines (interleukin 1 [IL-1], tumor necrosis factor alpha [TNF-alpha], interferon-gamma [INF-gamma], and IL-2) and performed immunohistochemistry. RESULTS: Arterial oxygenation was greatest in the recipient group transfected with TGF-beta1-active at 24 hours before transplantation compared with CAT, TGF-beta1-latent, and 18-hour cold ischemia groups (p < 0.01). The W/D ratio and myeloperoxidase decreased in both 24- and 48-hour groups, with TGF-beta1-active compared with CAT, and 18-hour cold ischemia groups (W/D, p < 0.02 and p < 0.004, respectively; myeloperoxidase, p < 0.05 and p < 0.01, respectively). All pro-inflammatory cytokines decreased in the 24-hour TGF-beta1-active group compared with CAT, TGF-beta1-latent, 18-hour and 1-hour cold ischemia, and non-treated lung groups (IL-1beta, p < 0.03; TNF-alpha, p < 0.02; IFN-gamma, p < 0.001; IL-2, p < 0.0001). In 24- and 48-hour groups with TGF-beta1-active, immunohistochemistry showed marked staining of Type I and Type II alveolar cells and of macrophages from the apical to the caudal sections of the lung grafts. CONCLUSIONS: Recipient IM administration of naked plasmid encoding TGF-beta1-active before transplantation ameliorates lung isograft reperfusion injury after prolonged ischemia.     

Adenovirus mediated gene transfer into rat lung grafts at the time of harvest

Objective: New methods to introduce genetic material into cells in vivo may revolutionize current treatment modalities. Expression of functional genes in lung allografts could be used as a prophylactic strategy for reperfusion injury and rejection. We studied the feasibility of ex vivo adenovirus mediated transfection of rat lung allografts. Methods: In group I (n=3) donor rat lungs (Fisher) were flushed with Low Potassium Dextran Glucose (LPDG) solution (20 ml, 20°C). 4×10¹¹ viral particles of adenovirus 5 containing the E. coli lacZ reporter gene coding for β-galactosidase (AdCMV-β-Gal) were added to the last mililiter of the flush solution. Lung grafts were stored for 3.5 h at room temperature followed by syngenic orthotopic transplantation (Fisher to Fisher) using a microsurgical cuff technique. On postoperative day 5 the heart lung block was extracted and flushed with x-Gal (β-Gal substrate) and kept in x-Gal for 3 h at 37°C. Color development was observed macroscopically and plastic embedded sections were used for histologic examination. Group II grafts (n=3) served as controls and were flushed without adenovirus. Results: X-Gal stained the transfected lung grafts blue, indicating high reporter gene expression. Control lungs did not stain with x-Gal. In group I histological examination demonstrated transfection predominantly in type II pneumocytes. Surprisingly endothelial cells showed no β-Gal activity. Conclusion: This study demonstrates that ex vivo transfection of lung grafts at the time of harvest is a feasible method of gene transfer and results in gene expression after transplantation.     

In vivo adenovirus-mediated endothelial nitric oxide synthase gene transfer ameliorates lung allograft ischemia-reperfusion injury

OBJECTIVE: Nitric oxide regulates vascular tone, inhibits platelet aggregation, and inhibits leukocyte adhesion, all of which are important modulators of ischemia-reperfusion injury. This study aimed to determine the effects of endothelial constitutive nitric oxide synthase gene transfer on ischemia-reperfusion injury in a rat lung transplant model. METHODS: In group I, donor animals were injected intravenously with 5 x 10(9) pfu of adenovirus-encoding endothelial constitutive nitric oxide synthase. Groups II and III served as controls, whereby donor animals were injected with either 5 x 10(9) pfu of adenovirus encoding beta-galactosidase or saline solution, respectively. Twenty-four hours after injection, left lungs were harvested and preserved for 18 hours at 4 degrees C, then implanted into isogeneic recipients, which were put to death 24 hours later. Recombinant endothelial constitutive nitric oxide synthase gene expression was evaluated by Western blotting and immunohistochemistry. Lung grafts were assessed by measuring arterial oxygenation, myeloperoxidase activity, and wet/dry weight ratios. RESULTS: Western blotting confirmed the overexpression of endothelial constitutive nitric oxide synthase in lungs so transfected compared with controls. Twenty-four hours after reperfusion, mean arterial oxygenation was significantly improved in group I compared with group II and III controls (189.4 +/- 47.1 mm Hg vs 71.7 +/- 8.9 mm Hg and 67.8 +/- 12.2 mm Hg, P =.02, P =.01, respectively). Myeloperoxidase activity, a reflection of tissue neutrophil sequestration, was also significantly reduced in group I compared with groups II and III (0.136 +/- 0.038 DeltaOD/mg/min vs 0. 587 +/- 0.077 and 0.489 +/- 0.126 DeltaOD/mg/min, P =.001, P =.01, respectively). CONCLUSION: Adenovirus-mediated gene transfer with endothelial constitutive nitric oxide synthase ameliorates ischemia-reperfusion injury as manifested by significantly improved oxygenation and decreased neutrophil sequestration in transplanted lung isografts. Endothelial constitutive nitric oxide synthase gene transfer may reduce acute lung dysfunction after lung transplantation.     

In Vivo Molecular Imaging Characterizes Pulmonary Gene Expression During Experimental Lung Transplantation

Experimental gene therapy is a promising strategy to prevent ischemia-reperfusion (I/R) injury and allograft rejection after lung transplantation, and methods will eventually be needed to characterize pulmonary transgene expression in vivo in humans. Therefore, we studied positron emission tomography (PET) as a means of performing in vivo molecular imaging in rodent models of lung transplantation. Rats were transfected endotracheally with adenovirus encoding a fusion gene of a mutant Herpes simplex virus-1 thymidine kinase and the green fluorescent protein gene (the former serving as an imaging reporter gene). Twenty-four hours after transfection, lungs were transplanted in groups representing normal transplantation, I/R injury and acute allograft rejection. Imaging was obtained either 24 h after transplantation to study reperfusion injury or 4 days after transplantation to study graft rejection. After imaging, lungs were excised and analyzed for thymidine kinase activity. Imaging detected transgene expression in transplanted lungs even in the presence of acute rejection or I/R injury. The PET imaging signal correlated with in vitro lung tissue assays of thymidine kinase activity (r(2) = 0.534). Thus, noninvasive molecular imaging with PET is a feasible, sensitive and quantitative method for characterizing pulmonary transgene expression in experimental lung transplantation.     

Interleukin-1 receptor antagonist as a biomarker for bronchiolitis obliterans syndrome in lung transplant recipients

BACKGROUND: The major limitation to survival after lung transplantation is bronchiolitis obliterative syndrome (BOS). BOS is a chronic inflammatory/immunologic process characterized by fibroproliferation, matrix deposition, and obliteration of the airways. The mechanism(s) that lead to fibro-obliteration of allograft airways have not been fully elucidated. Interleukin-1 receptor antagonist (IL-1Ra) is a naturally occurring antagonist of the pro-inflammatory cytokine IL-1 and has been associated with a number of fibroproliferative diseases. METHODS: We determined whether IL-1Ra, as compared to IL-1beta, IL-10, transforming growth factor (TGF)-beta, and tumor necrosis factor (TNF)-alpha, in the bronchoalveolar lavage fluid (BALF) from lung transplant recipients was associated with BOS. BALF was collected from three groups of patients: BOS (n=22), acute rejection (n=33), and healthy transplant recipients (n=30). RESULTS: IL-1Ra levels were significantly elevated in patients with BOS compared to healthy lung transplant recipients and patients with acute rejection (P<0.001 and P<0.05, respectively). Furthermore, when patients with BOS had their BALF analyzed from their last bronchoscopy before the development of BOS (Future BOS [FBOS] group) (n=20), their levels of IL-1Ra were also significantly elevated compared to healthy lung transplant recipients and patients with acute rejection (P<0.001 and P<0.05, respectively). Importantly, the elevated levels of IL-1Ra in the BOS and FBOS groups were not accompanied by any significant increases in IL-1beta, IL-10, TGF-beta, or TNF-alpha. CONCLUSION: These findings suggest that elevated levels of IL-1Ra may be attenuating IL-1 bioactivity during the pathogenesis of BOS and creating a local environment that favors fibroproliferation and matrix deposition.     

Endobronchial transfection of naked TGF-beta1 cDNA attenuates acute lung rejection.

BACKGROUND: We investigated endobronchial transfection of CAT and TGF-beta1 cDNA selectively delivered to the lung graft with or without liposomes. METHODS: Phase I: F344 rats received 130 microg of naked plasmid pCF1-CAT or complexed to liposome GL67 via left main bronchus instillation. Rats were awakened (pCF1-CAT, n = 4; GL67:pCF1-CAT, n = 4) or served as donors in an isogenic transplant (pCF1-CAT, n = 5; GL67:pCF1-CAT, n = 5). ELISA was performed on lungs, hearts, and livers on POD 2. Phase II: BN lungs received TGF-beta1 sense (n = 6); antisense (n = 5); GL67:TGF-beta1 sense (n = 10); or saline solution (n = 10). F344 recipients were sacrificed on POD 5. The arterial pO2 and rejection were assessed. RT-PCR for murine TGF-beta1 was performed. RESULTS: Phase I: CAT expression was 519+/-287 pg and 63+/-68 with pCF1-CAT and 104+/-67 and 37+/-45 with GL67:pCF1-CAT, respectively, in the non-transplant and in the transplant setting. No protein was detected in the hearts, livers, and in the native lung of the recipients. Phase II: RT-PCR confirmed murine TGF-beta1 transfection. pO2 was 362.7+/-110.2 (mean mm Hg +/- SD) for sense TGF-beta1; 146.88+/-85.5 for antisense; 241.5+/-181.5 for GL67-TGF-beta1 sense; and 88.4+/-38.7 for saline. TGF-beta1 sense versus all other groups, p<0.05, GL67-TGF-beta1 sense versus saline, p = 0.01. Rejection was significantly lower for TGF-beta1 sense versus saline, p = 0.04. CONCLUSIONS: Endobronchial administration of naked plasmid achieves selective transfection of lung grafts. Using this strategy, TGF-beta1 reduces early lung allograft rejection.     

腺病毒介导TGF-β 1转基因联合FTY720对大鼠同种异体肺移植缺血-再灌注损伤的保护作用

目的观察腺病毒介导TGF-β₁转基因联合FTY720对大鼠同种异体肺移植缺血-再灌注损伤(IRI)的影响。 方法采用改良的三袖套法吻合技术建立大鼠左肺原位移植模型。以SD大鼠为供受体,随机分为假手术对照组、空白对照组、空载体对照组、TGF-β₁组、FTY720组和TGF-β₁＋FTY720组,每组6只。移植肺再灌注后4 h,检测各项生理指标。 结果腺病毒介导TGF-β₁转基因组荧光现象显著,并确定1×10¹⁰ PFU TGF-β₁基因腺病毒载体为较优转染浓度。肺移植再灌注后4 h,与假手术对照组比较,空白对照组和空载体对照组出现典型的移植肺IRI症状,肺动脉血氧分压(PaO₂)明显降低,二氧化碳分压(PaCO₂)和湿/干重(W/D)比值明显升高,超氧化物歧化酶(SOD)活性明显降低,丙二醛(MDA)含量、髓过氧化物酶(MPO)活性和肿瘤坏死因子-α(TNF-α)含量明显升高。与空白对照组和空载体对照组比较,TGF-β₁组、FTY720组和TGF-β₁＋FTY720组移植肺组织水肿和间质炎症等病理组织学现象明显减轻,PaO₂明显上升,PaCO₂和W/D比值明显下降,SOD活性明显增加,MDA含量、MPO活性和TNF-α含量明显降低。其中,联合处理组病理症状表现出更为明显的减轻趋势。 结论腺病毒介导TGF-β₁转基因联合FTY720能有效减轻大鼠同种异体肺移植IRI。     

DNA-liposome versus adenoviral mediated gene transfer of transforming growth factor beta1 in vascularized cardiac allografts: differential sensitivity of CD4+ and CD8+ T cells to transforming growth factor beta1

We have developed a model of transforming growth factor (TGF)beta1 gene transfer into mouse vascularized cardiac allografts to study the use of gene transfer as an immunosuppressive therapy in transplantation. Donor hearts were perfused with either DNA-liposome complexes or adenoviral vectors that encode the active form of human TGFbeta1. DNA-liposome mediated transfection prolonged allograft survival in approximately two-thirds of transplant recipients, while adenoviral delivery of TGFbeta1 was not protective. Protective TGFbeta1 gene transfer was associated with reduced Th1 responses and an inhibition of the alloantibody isotype switch. The protective effects of TGFbeta1 gene transfer were overridden by exogenous interleukin-12 administration. Interestingly, alloreactive CD4+ and CD8+ cells exhibited distinct sensitivities to TGFbeta1 gene transfer: CD4+ Th1 function was abrogated by this modality, although CD8+ Th1 function was not. Transient depletion of recipient CD8+ cells markedly prolonged the survival of grafts transfected with either DNA-liposome complexes or adenoviral vectors. Transgene expression persisted for at least 60 days, and Th1 responses were not detectable until CD8+ T cells repopulated the periphery. However, long-term transfected allografts appeared to exhibit exacerbated fibrosis and neointimal development. These manifestations of chronic rejection were absent in long-term transfected isografts, suggesting that long-term expression of active TGFbeta1 alone is not sufficient to induce fibrosis of the grafts. Collectively, these data illustrate the utility of immunosuppressive gene therapy as a treatment for transplantation when combined with additional conditioning regimens. Further, they illustrate that alloreactive CD4+ and CD8+ cells may be differentially influenced by cytokine manipulation strategies.